Deficiencies in propionyl-CoA carboxylase (PCC) precipitate life- threatening propionic acidemia in humans together with mental retardation. PCC is encoded by two genes, PCCA and PCCB, on separate chromosomes, and exhibits complex complementation patterns in compound heterozygotes. 58 separate mutations in PCCA and PCCB genes have been identified. Four of the beta mutations occur within a conserved region in exon 12 and within the same 14 nucleotides in 21 out of 50 affected alleles. To date only two betaPCC mutations have been expressed in E. coli and characterized. Nothing is known about the region of either gene. Additionally, neither the ligand binding sites nor the tertiary structure of the enzyme have been identified. These experiments are designed to improve our understanding of the biochemistry, genetics, and molecular cell biology of inborn mutations of PCC. These studies are aimed at 1) determining the organization of both the PCCA and PCCB genes, defining the exon/intron boundaries and 5'- and 3'- flanking regions; 2) isolating and characterizing the human PCCA and PCCB promoters in our PCC genomic clones; 3) expressing cDNA constructs containing cDNA constructs containing the human alpha and beta PCC mutations in Escherichia coli and in human cells to characterize the mutations on mitochondrial import, assembly, and catalytic properties of the recombinant protein; 4) ascertaining the ATP and propionyl CoA binding motifs in the enzyme structure; 5) containing to optimize the expression, purification, and the crystallization conditions for techniques for generating PCC crystals suitable for solving the structure of the enzyme by X-ray diffraction. Specific techniques employed will include: cloning and expressing both subunits of the enzyme in bacterial and human cells; protein purification techniques; preparation of RNA and genomic DNA; Southern, Northern and Western blots; DNA sequencing, and crystallography. The major aim of this project is to elucidate the tertiary and quaternary structure of the enzyme and the role of mutations in disabling PCC. These studies will improve the genotype/phenotype correlations leading to improved therapeutic rationales.